Research in the Cell Biology Section, Neurogenetics Branch focuses on the molecular mechanisms underlying a number of neurodegenerative disorders, including mitochondrial disorders, dystonia, and the hereditary spastic paraplegias (HSPs). These disorders, which together afflict millions of Americans, worsen insidiously over a number of years, and treatment options are limited for many of them. Our laboratory is investigating inherited forms of these disorders, using molecular and cell biology approaches to study how mutations in disease genes ultimately result in cellular dysfunction. In this project, we are focusing on the HSPs. One major research theme involves the characterization and functional analysis of the hereditary spastic paraplegia type 3A (SPG3A) protein, atlastin-1. In 2009, we reported in the journal Cell that atlastin-1 is a member of a ubiquitous family of GTPases that interact with two families of ER shaping proteins to generate the tubular endoplasmic reticulum (ER) network. Interestingly, atlastin-1 interacts with the SPG31 protein REEP1, which is an ER shaping protein, as well as the SPG4 protein spastin, a microtubule-severing ATPase. In 2010, we published a study in the Journal of Clinical Investigation demonstrating that these three proteins interact with one another to organize the tubular ER network in conjunction with the microtubule cytoskeleton. Since SPG3A, SPG4, and SGP31 account for well over 50% of all HSP cases, we suggest ER network defects as the predominant neuropathologic mechanism for the HSPs. This is supported by the recent identification of numerous other HSP proteins that regulate ER morphology, including CPT1C protein (in collaboration with Dr. Kurt Fischbeck). Over the past year, we have continued to develop animal models for SPG31 (knock out) and SPG3A (knockout and knock in) to evaluate the extent of ER morphology changes using both in vivo and ex vivo studies. We are employing both high-throughput electron microscopy (in collaboration with Dr. Mark Terasaki) and super-resolution confocal microscopy to examine the changes in tubular ER within neuronal axons in response to these genetic manipulations. In addition, we have identified interactions of these proteins with several other proteins mutated in the HSPs, expanding the number of HSP cases related to defects in ER network formation. Furthermore, we are actively generating in situ models for many of the HSPs through the production of patient-derived, induced pluripotent stem cells that are then differentiated into telecephalic neurons, in collaboration with Dr. Xue-Jun Li. A number of these studies were published in 2014 in the journals Stem Cells and Human Molecular Genetics. ER morphology and dynamics in these and other cells are being evaluated using a number of emerging super-resolution microscopy techniques in collaboration with Drs. Jennifer Lippincott-Schwartz, Eric Betzig, and Harald Hess. Finally, we are working with Dr. Niamh O'Sullivan on fly models of these HSPs. A key aspect of ER function possibly related to disease pathogenesis is the formation of lipid droplets, and this is an area of emphasis for our cellular and organismal studies. We are in the process of completing several studies tying changes in ER morphology to alterations in lipid droplet biogenesis. As part of these studies, we have used CRISPR technologies to knock out all three atlastin isoforms from cell lines such as HeLa and NIH-3T3. We have also utilized advanced imaging techniques such as CT scans and MR spectroscopy to study changes in fat tissue in HSP mouse models non-invasively; one such study was published in Hum Mol Genet in 2016. These data were used for the planning of a large clinical natural history trial in patients with the three most common forms of autosomal dominant HSP (SPG4, SPG3A, and SPG31), which has just begun recruiting. Taken together, we expect that our studies will advance our understanding of the molecular pathogenesis of the HSPs. Such an understanding at the molecular and cellular levels will hopefully lead to novel treatments to prevent the progression of these disorders.